PROJECT SUMMARY Ewing Sarcoma (EWS) is the second most common primary bone cancer and a deadly threat to US children and adolescents. Despite great efforts over the last few decades, early detection of pediatric EWS is still challenging for standard imaging and cytogenetic modalities and very little improvement in long-term survival of EWS has been achieved. Patients initially presenting without clinically overt metastases at diagnosis have a 5- year overall survival of 70-75%; in contrast, the 5-year overall survival for metastatic EWS is below 30% due to high prevalence of disease recurrence/relapse. The long-term survival is 22% to 24% for patients with limited localized relapse and even lower for distant relapse. Tissue biopsy for pathological or cytological diagnosis and monitoring of EWS is extremely invasive and difficult, especially in young children. The lack of reliable blood biomarkers presents a serious obstacle to the treatment and management of EWS, more importantly, recurrent or metastatic EWS. Thus novel diagnostic and prognostic biomarkers are urgently needed to improve clinical outcome of this deadly pediatric cancer. Circulating exosomes are emerging as a new paradigm of ?liquid biopsy? for non-invasive cancer diagnosis and monitoring. Exosomes are small membrane vesicles of 30-150 nm in size secreted by most cells. Growing evidence has shown important biological roles and clinical relevance of exosomes. In various cancers, tumor-derived exosomes have been found to be accumulated in human biofluids, such as blood, and enriched with a set of biomolecules from the cells of origin, such as proteins and RNAs, which may constitute a ?cancer signature?. However, biology and clinical value of exosomes remain largely unknown, due in part to the challenges in isolation and analysis of such small, dynamic and molecularly diverse vesicles. Very few, if any, exosome studies in EWS have been reported. To address this obvious gap in both analytical technologies and the precision medicine for EWS, we propose to develop and validate the nanotechnology-inspired, integrated microfluidic platforms that offer unprecedented analytical capabilities for measuring protein and mRNA markers in circulating exosomes derived from EWS. These new technologies will be validated to profile circulating exosomes in a unique longitudinal series of blood samples from pediatric EWS patients at initial diagnosis, during therapy and off therapy, aiming to identify exosomal markers that can track therapeutic response and detect early disease recurrence. While designed to specifically target EWS as proof-of-principle, this project will ultimately provide transformative platform technologies for basic and clinical investigation of a wide range of malignancies and diseases.